Background: As a ubiquitous apicomplexan parasite, Toxoplasma gondii causes huge economic losses and poses a great threat to the health of animals, including humans, worldwide. In some kangaroo species, T. gondii can be fatal. To date, little information is available on T. gondii infection in the red kangaroos in east China. At a zoo in east China, thirteen red kangaroos consecutively developed clinical signs from July to November 2016, resulting in the deaths of seven, three of which were analyzed in this study. Methods: In the present study, ascitic fluid, blood and samples from the brain, heart, liver, spleen, lung, kidney, and mesenteric lymph nodes of three dead red kangaroos were collected. The pathogen was explored through microscopic observation, nested PCR, immunofluorescence antibody test (IFAT), hematoxylin–eosin (HE) staining, and immunohistochemistry (IHC) staining, respectively. Meanwhile, the potential source of the infection was also investigated by testing the blood of stray cats in the zoo for T. gondii using nested PCR. Results: Three dead red kangaroos were subjected to a necropsy, and organisms resembling T. gondii were detected in their ascitic fluids under microscope. This infection was further confirmed by a nested PCR assay, which resulted in a successful amplification and sequencing of the 433 bp fragment of the T. gondii 5.8S rRNA gene in all the dissected tissues, including heart, liver, spleen, lung, kidney, lymph nodes, cecum, and brain, as well as in body fluids (blood and ascitic fluid). Furthermore, the tachyzoites were observed in the heart, liver, spleen, lymph nodes, cecum, and brain through IFAT and HE staining. Administration of classic drugs (sulfadiazine and pyrimethamine) against T. gondii significantly alleviated the clinical signs of the sick kangaroos. The possible source of this infection was traced to a native stray cat, as T. gondii DNA was detected in its blood. Conclusions: In the present study, lethal T. gondii infection in red kangaroos has been described for the first time in east China, highlighting the necessity and urgency for close and long-term surveillance of this parasite infection in captive animals. The same strain of T. gondii detected in kangaroos as that found in stray cats wandering in the same area emphasizes the importance of controlling stray cat populations to mitigate the risk of Toxoplasma transmission to other animals.

Cholera caused by Vibrio cholerae remains a major public health concern in many countries. The greatest obstacle to detection of V. cholerae contamination in drinking water or aquatic environments mainly relates to sample preparation steps, especially the enrichment step. In this study, immunomagnetic separation methods were developed based on sequence-defined recombinant antibodies (rAbs) against V. cholerae, then used for the specific and efficient enrichment of V. cholerae in water samples. Using the variable region genes of the anti-V. cholerae monoclonal antibodies (mAbs) 5F2, the full-length IgG rAbs (R5F2) were produced using mammalian human embryonic kidney 293T cells. Two antibodies, 5F2 and R5F2, were used to prepare immunomagnetic beads (IMBs), and their capture efficiencies (CEs) were evaluated. The results showed that 0.4 mg of 5F2-IMBs and R5F2-IMBs exhibited good CEs (96.0% and 75.9%, respectively) against V. cholerae within 40 min. The IMBs could still effectively capture V. cholerae in large-volume reaction systems (5 ml to 25 ml). The CEs of 5F2-IMBs and R5F2-IMBs ranged from 90.2% to 70.7% and 65.1% to 44.2%, respectively. Furthermore, 5F2-IMBs and R5F2-IMBs did not show significant cross-reactivity with other bacteria and exhibited high specificity. When R5F2-IMS was used in combination with quantitative real-time PCR, the detection limit was approximately 5 colony-forming units/25 ml after enrichment for 4 h. Our results suggest that the rAbs produced herein could provide useful alternatives to traditional hybridoma-based antibodies for accurate detection of V. cholerae in food safety and environmental monitoring.

Type VI secretion system 2 (T6SS2) of Vibrio parahaemolyticus is required for cell adhesion and autophagy in macrophages; however, other phenotypes conferred by this T6SS have not been thoroughly investigated. We deleted TssL2, a key component of T6SS2 assembly, to explore the role of the T6SS2 in environmental adaptation and virulence. TssL2 deletion reduced Hcp2 secretion, suggesting that TssL2 played an important role in activity of functional T6SS2. We found that TssL2 was necessary for cell aggregation, wrinkly phenotype formation, and participates in motility and biofilm formation by regulating related genes, suggesting that TssL2 was essential for V. parahaemolyticus to adapt changing environments. In addition, this study demonstrated TssL2 significantly affected adhesion, cytotoxicity, bacterial colonization ability, and mortality in mice, even the levels of the proinflammatory cytokines IL-6 and IL-8, suggesting that TssL2 was involved in bacterial virulence and immunity. Proteome analysis revealed that TssL2 significantly affected the expression of 163 proteins related to ABC transporter systems, flagellar assembly, biofilm formation, and multiple microbial metabolism pathways, some of which supported the effect of TssL2 on the different phenotypes of V. parahaemolyticus. Among them, the decreased expression of the T3SS1 and T2SS proteins was confirmed by the results of gene transcription, which may be the main reason for the decrease in cytotoxicity. Altogether, these findings further our understanding of T6SS2 components on environmental adaption and virulence during bacterial infection. • The role of T6SS2 in V. parahaemolyticus was far from clear. • TssL2 participates in cell aggregation, wrinkly phenotype formation, motility, and biofilm formation. • TssL2 is essential for cell bacterial colonization, cytotoxicity, virulence, and proinflammatory cytokine production.

Toxoplasma gondii (T. gondii) is an important zoonotic pathogen which induces both acute and chronic toxoplasmosis. Timely diagnosis of T. gondii is crucial for effective disease management. Here, we present a pioneering approach using europium (III)-chelated nanoparticles (EuNPs) in a rapid lateral flow immunochromatographic test strip (ICTS) for detecting T. gondii antibodies in serum samples. By conjugating EuNPs with Staphylococcus aureus protein A, we efficiently captured T. gondii-specific antibodies, which bound to T. gondii antigens on the test line (T-line), generating a distinct fluorescent signal. Employing this novel method, we conducted an extensive epidemiological investigation of T. gondii infections among dogs and cats in Shanghai, China. This innovative ICTS allows for rapid results within 25 min, which include a qualitative result through naked-eye observation under an ultraviolet lamp and a quantitative one derived using a strip reader. With a detection limit of 1:6400 for dog positive serum and no cross-reactivity with other canine and feline pathogens, the EuNPs-ICTS demonstrated excellent consistency with standard enzyme-linked immunosorbent assay results for dogs (κ = 0.91) and cats (κ = 0.92). In addition, 20.38% of 996 dog serum samples and 14.18% of 416 cat serum samples revealed T. gondii antibodies, highlighting the efficacy of this approach. Our study presents a rapid, sensitive, specific, and reproducible EuNPs-ICTS, serving as a promising tool for on-the-spot diagnosis of T. gondii infections in dogs and cats.

免疫磁珠技术操作简单、快速高效,目前已广泛应用于细菌的快速分离纯化等领域,本研究旨在建立一种捕获率高、特异性好的单增李斯特菌免疫磁珠分离方法.首先通过杂交瘤技术制备出 8 株抗单增李斯特菌单克隆抗体,效价均达到 1 ∶ 1024000,且特异性较好.利用效价最高的腹水6-E6 制备单增李斯特菌免疫磁珠,并对单增李斯特菌免疫磁珠最适条件进行优化,结果选择MEST(7.0)为偶联缓冲液、磁珠直径为750 nm、免疫磁珠添加量为0.4 mg、捕获时间45 min、单增李斯特菌含量在1×104 CFU/mL时,免疫磁珠捕获率最高可达76.29%.在模拟牛奶样品中免疫磁珠特异性捕获率可达到 67.71%.该分离方法可快速且特异地分离单增李斯特菌,操作简便,为快速分离食品中的单增李斯特菌并实现快速检测提供了技术支持.

链球菌是引起奶牛乳房炎的重要病原菌,其中无乳链球菌(S.agalactiae)、停乳链球菌(S.dysgalac-tiae)和乳房链球菌(S.uberis)是奶牛乳房炎主要的致病性链球菌.为同时、快速地检测牛奶样品中这3种重要链球菌,本研究根据无乳链球菌Sip基因、停乳链球菌Mig基因及乳房链球菌pauA基因设计特异性引物,同时以细菌16SrRNA基因为内参,建立了多重PCR检测方法,并进行条件优化.结果显示,该方法最佳退火温度为58℃,最佳扩增循环数为25个循环,本方法同时检测无乳链球菌、停乳链球菌及乳房链球菌时,检测限分别为1×103 CFU/mL、1×103 CFU/mL及1×104 CFU/mL,灵敏性高.该方法对大肠杆菌、金黄色葡萄球菌、肺炎克雷伯氏菌、单核细胞增生李斯特菌等10种菌均无交叉反应,特异性好.模拟样品结果显示,当奶样中3种菌的初始浓度均为1 CFU/mL时,37℃增菌6 h即可检出乳房链球菌,增菌8 h以上3种菌可全部检出.结果表明,该多重PCR检测方法具有较好的特异性和灵敏性,对同时快速检测牛奶中无乳链球菌、停乳链球菌及乳房链球菌具有重要意义,也为奶牛乳房炎的监管、诊治提供了技术支持.

组氨酸氨解酶(HutH)作为组氨酸代谢通路上的第一个代谢酶,控制细菌内部组氨酸的代谢.HutH在多数细菌中高度保守,参与细菌的能量代谢平衡.[目的]选取HutH作为研究对象,探究其对副溶血弧菌生物学特性以及致病性的影响.[方法]利用同源重组的方法构建缺失株Δ hutH和回补株CΔhutH.研究HutH对副溶血弧菌生长特性、组氨酸利用能力、组氨酸代谢相关基因表达水平、运动性、生物被膜、环境耐受、细胞毒性以及对小鼠毒力的影响.[结果]与野生型菌株相比,hutH基因缺失不影响副溶血弧菌的生长特性、耐酸耐碱能力、耐盐能力和群集运动.但ΔhutH在组氨酸作为唯一碳源的M9极限培养基中生长受到显著抑制.另外,我们证实,hutH基因缺失使组氨酸代谢操纵子内的相关基因转录水平显著下降,基因VP0889的表达水平提高.hutH基因缺失导致副溶血弧菌生物被膜形成能力下降、泳动能力下降、对HeLa细胞的毒性降低、对ICR小鼠的致死率显著降低.[结论]本研究表明,hutH基因缺失影响副溶血弧菌代谢

pepT基因编码一种金属依赖性肽酶T(peptidase T,PepT),能特异性催化三肽N端氨基酸,因此也称为氨肽酶T.研究发现大多数氨肽酶参与细菌蛋白质新陈代谢和调节三肽活性,但关于PepT在细菌毒力及致病性方面的报道较少.[目的]本文选取PepT为研究对象,研究其对副溶血弧菌生物学特性及致病性的影响.[方法]通过构建缺失株ΔpepT和回补株CΔpepT,比较菌株在运动性、生物被膜、环境耐受、细胞毒性等方面的差异.[结果]与野生株相比,ΔpepT缺失株的极性鞭毛转录水平极显著下降,浮游运动能力降低;同时生物被膜形成能力减弱,而细菌群集运动及环境耐受能力无显著差异.此外,缺失pepT基因会导致副溶血弧菌的细胞毒性和小鼠毒力作用显著下降.[结论]pepT基因与副溶血弧菌浮游运动和生物被膜形成能力相关,并且影响其致病性.

本研究以灭活阪崎克罗诺杆菌(CS)为免疫原免疫BALB/c小鼠,5次免疫后取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合.利用间接ELISA法筛选阳性杂交瘤细胞株,制备抗CS特异性单克隆抗体.对单克隆抗体进行效价及特异性测定,确定其抗体亚型及单克隆抗体之间的抗原竞争性.结果表明:经4次亚克隆后得到2株稳定产生抗体的细胞株,分别命名为9D10、2H2,9D10抗体和2H2抗体效价较高,分别为2×106、6×106;9D10抗体和2H2抗体与单增李斯特菌、大肠杆菌、鼠伤寒沙门菌等均不发生交叉反应,特异性良好;2株抗体重链均为IgG2a,轻链均为Kappa;2株抗体之间无抗原竞争性.抗CS单克隆抗体的制备可为其快速检测方法的建立提供基础.

以阪崎克罗诺杆菌特异性基因16SrRNA和外膜蛋白基因ompA为靶序列,设计适用于重组酶聚合酶扩增-侧流层析技术(RPA-LF)的引物和探针,并优化反应条件.结果表明,RPA反应可在25～45℃进行,10～20 min即可完成.优化条件后,RPA-LF总反应时间仅为20 min,包括RPA在39℃反应15 min,RPA反应产物在LF试纸条上反应5 min,即可用肉眼判断结果.该方法具有较高的灵敏度,最低检测限可以达到80 pg/反应,约为960 CFU/反应.与7种常见的食源性病原菌都没有交叉反应,表明该方法具有较好的特异性.此外,该方法能够同时识别6种阪崎克罗诺杆菌同属菌,表明该方法广谱性好.本研究中建立的针对阪崎克罗诺杆菌的RPA-LF检测方法,具有操作简便、快速敏感的优点,为该菌的现场快速检测打下了基础.

[目的]探究OmpR在副溶血弧菌生物学特性和致病性中发挥的作用.[方法]利用同源重组技术构建了副溶血弧菌ompR基因缺失株(ΔompR)和互补株(CΔompR),分析各菌株的生长特性、运动性和生物被膜形成能力的差异;比较各菌株对细胞黏附、细胞毒性和小鼠致病性的影响.[结果]ompR基因缺失对副溶血弧菌的生长特性、运动性以及细胞毒性无显著影响.但与野生株相比,ΔompR的生物被膜的形成能力显著降低;感染ΔompR的小鼠存活率升高了 25％,病变程度更低;ΔompR在小鼠心脏、肝脏和肾脏中的载菌量显著低于野生株,互补株毒力基本恢复至野生株水平.[结论]OmpR参与副溶血弧菌生物被膜形成和致病过程,是副溶血弧菌潜在的毒力因子.

Vibrio parahaemolyticus is a marine pathogen thought to be the leading cause of seafood-borne gastroenteritis globally, urgently requiring efficient management methods. V. parahaemolyticus encodes 12 resistance/nodulation/division (RND) efflux systems. However, research on these systems is still in its infancy. In this study, we discovered that the inactivation of VmeL, a membrane fusion protein within the RND efflux systems, led to reduction of the ability of biofilm formation. Further results displayed that the decreased capacity of Congo red binding and the colony of ΔvmeL is more translucent compared with wild type strains, suggested reduced biofilm formation due to decreased production of biofilm exopolysaccharide upon vmeL deletion. In addition, the deletion of vmeL abolished surface swarming and swimming motility of V. parahaemolyticus. Additionally, deletion of vmeL weakened the cytotoxicity of V. parahaemolyticus towards HeLa cells, and impaired its virulence in a murine intraperitoneal infection assay. Finally, through RNA-sequencing, we ascertained that there were 716 upregulated genes and 247 downregulated genes in ΔvmeL strain. KEGG enrichment analysis revealed that quorum sensing, bacterial secretion systems, ATP-binding cassette transporters, and various amino acid metabolism pathways were altered due to the inactivation of vmeL. qRT-PCR further confirmed that genes accountable to the type III secretion system (T3SS1) and lateral flagella were negatively affected by vmeL deletion. Taken together, our results suggest that VmeL plays an important role in pathogenicity, making it a good target for managing infection with V. parahaemolyticus.

建立一种能够快速检测动物源性食品中多种氟喹诺酮类药物残留的直接竞争化学发光酶免疫法.用诺氟沙星(NOR)分子修饰后制备的完全抗原免疫小鼠,获得对10种氟喹诺酮类药物具有广谱特异性的单克隆抗体,采用二因子交叉试验确定包被原与酶标抗体的最佳稀释度,通过单因素试验优化最佳包被条件、封闭液种类、竞争反应时间,从而建立反应过程的最佳条件,并对方法的灵敏度、交叉反应率、精密度进行评价.结果表明:NOR-1-OVA包被浓度0.3μg/mL,酶标抗体的稀释倍数为1:8000时为最佳稀释浓度;4℃包被过夜,封闭液为1％BSA,竞争时间为1 h时为最优反应条件.对10种氟喹诺酮类药物的灵敏度(IC50)为1.46～11.57 ng/mL,交叉反应率为12.6％～118.9％,并且与其他非喹诺酮类药物无交叉反应率;检出限为0.07～0.27 ng/mL,且批内差异和批间差异均低于10％;加标回收率为73.3％～103.1％,变异系数为1.9％～11.8％.以上结果表明,该方法可作为动物源性食品中10种FQs残留检测的常规方法.

为建立奶制品中单核细胞增生李斯特菌(Listeria monocytogenes,LM)的快速检测方法,分别制备抗LM的单克隆抗体和家兔多克隆抗体,以方阵滴定法确定最佳单抗包被浓度和多抗工作浓度,从而建立LM的双抗体夹心酶联免疫吸附试验(ELISA)检测方法.结果 表明,抗LM的单抗和多抗效价分别为1∶128000和1∶2560000,分别以抗LM单抗和多抗作为捕获抗体与检测抗体;经优化后选择抗LM单克隆抗体稀释至1∶1000、抗LM兔多抗稀释至1∶4000建立ELISA检测方法.结果 显示:所建立的双抗体夹心ELISA方法对LM的最低检测限可达到1×105 CFU/mL,与常见的10种食源性病原微生物均无交叉反应,表明该方法的特异性良好.重复性试验结果显示,该方法的批内、批间变异系数均小于10％,表明该方法具有良好的重复性.模拟样品试验结果表明,在牛奶样本中添加LM至细菌浓度为1×100 CFU/mL时,增菌培养10h后用本方法即可检出阳性;牛奶样本的添加回收试验进一步表明回收率可达70.22％～91.59％,提示本方法可有效用于牛奶样本中LM的检测.综上,利用抗LM的单抗和兔多抗成功建立了双抗体夹心ELISA方法,该方法具有良好的敏感性、特异性和重复性,可为牛奶中LM的监测提供技术支撑.

为建立一种快速检测产志贺毒素大肠杆菌的基于侧流层析的重组酶聚合酶扩增技术(RPA-LF)方法,以志贺毒素stx1和stx2基因序列为靶序列,设计了用于RPA-LF方法的特异性引物及探针,并对RPA-LF反应条件进行优化.结果显示,针对stx1和stx2基因的最佳反应温度分别为39℃和37℃,最佳反应时间分别为10min和15min.最佳反应条件下,stxx1和stx2基因的最低检测限分别为1pg和10pg,灵敏度较高.该检测方法能够准确识别含stx1和/或stx2基因的大肠杆菌,且不与其他6种非目的病原菌发生交叉反应,表明本方法特异性良好.本研究建立的针对志贺毒素stx1和stx2基因的RPA-LF快速检测方法灵敏度和特异性较好,且检测时间短,结果直观可视.本试验为产志贺毒素大肠杆菌的筛选诊断和现场的快速检测开辟了 一个新的途径.

[目的]以副溶血弧菌VP2918为研究对象,研究其对副溶血弧菌的生物学特性和致病性的影响.[方法]利用同源重组技术构建了vp2918基因的基因缺失株(△vp2918)和互补株(C△vp2918),并对野生株、缺失株和互补株的细菌生长曲线、运动性、生物被膜形成能力、对HeLa细胞的黏附能力、细胞毒性、对小鼠的致死率和组织载菌量进行分析.[结果]缺失vp2918基因不影响副溶血弧菌的生长特性、运动性、生物被膜形成能力以及对HeLa细胞的黏附能力.但与野生株相比,△vp2918对HeLa细胞的毒性作用显著降低;感染△vp2918的小鼠症状明显减轻,存活率更高;△vp2918在小鼠脾脏和肝脏中的载菌量显著低于野生株,互补株毒力基本恢复至野生株水平.[结论]vp2918不参与副溶血弧菌的运动性和生物被膜形成能力等过程,但与该菌的致病性相关,为潜在的毒力因子.

Ⅵ型分泌系统(T6SS)是细菌的一种毒力因子分泌系统,通过分泌蛋白参与调控细菌的环境适应性和毒力.副溶血弧菌具有两个T6SS系统(T6SS1和T6SS2).[目的]前期通过差异蛋白质组学技术筛选到副溶血弧菌T6SS1相关的分泌蛋白,本文选择其中的VPA1500为研究对象,研究其基因缺失对副溶血弧菌的生物学特性及致病性的影响.[方法]利用同源重组技术构建缺失株ΔVPA1500和互补株CA VPA1500;分析各菌株生长特性、在体外的细菌竞争能力、运动性、细菌鞭毛相关基因的转录水平及生物被膜形成能力的差异,比较各菌株对细胞毒性、小鼠毒力、动物组织载菌量以及组织病理学变化的影响.[结果]与野生株相比,VPA1500基因缺失后不影响细菌的生长能力、生物被膜形成能力和群集运动,然而ΔVPA1500的浮泳运动能力显著下降;进一步通过透射电镜观察和实时定量PCR检测发现,VPA1500缺失影响副溶血弧菌鞭毛的形成;细菌竞争实验显示缺失VPA1500基因降低了副溶血弧菌野生株体外对大肠杆菌的杀伤能力;ΔVPA1500对细胞毒性、小鼠毒力以及在动物组织的定殖能力均显著低于野生株,互补株毒力基本恢复至野生株水平;组织病理学结果进一步表明,缺失VPA1500基因能够降低副溶血弧菌对小鼠组织的损伤.[结论]VPA1500参与副溶血弧菌的体外细菌竞争能力、浮游运动能力和致病性.

为建立一种快速、准确同时检测5种致病性弧菌的高通量液相芯片技术,本研究针对霍乱弧菌、副溶血弧菌、创伤弧菌、溶藻弧菌和拟态弧菌的10个目的基因,包括5个种特异性基因和5个毒力基因,分别设计特异性引物和探针,多重PCR扩增后的产物与偶联有不同核酸探针的微球混合物进行杂交反应,利用液相芯片检测仪分析结果.结果显示,本方法同时检测这5种致病性弧菌时,检出限最高达到1.8×102 CFU/mL;检测包括大肠杆菌、金黄色葡萄球菌、河流弧菌、沙门菌、李斯特杆菌和铜绿杆菌等常见细菌时,均无交叉反应,表明特异性好;检测不同批次的样品时,变异系数均小于8％,表明重复性良好.本研究建立的液相芯片检测方法能够快速同时检测这5种致病性弧菌,具有高通量、灵敏性高、重复性好、可控性强、节省样本和时间等优点,对水产品中多种重要致病性弧菌的检测和流行病学调查提供了技术支持.

为建立动物源性食品中沙拉沙星(SAR)残留的快速检测方法,本试验通过对盐酸沙拉沙星进行分子改造和偶联蛋白合成完全免疫原,免疫小鼠制备SAR单克隆抗体.通过棋盘法确定最佳包被原浓度及一抗稀释度,优化确定最佳反应条件,从而建立间接竞争化学发光酶联免疫(ic-CLEIA)检测方法,并通过灵敏度、精密度、交叉反应率、添加回收试验对该方法进行评价.紫外扫描结果显示,完全免疫原偶联成功;制备的SAR单克隆抗体效价达1 ∶ 4×106;包被原浓度为0.25 μg/mL,抗体稀释比为1:750 000,4℃包被过夜,5％脱脂乳封闭,竞争孵育1 h,酶标二抗稀释比为1:10 000,孵育1h为ic-CLEIA最佳反应条件;建立的ic-CLEIA方法标准曲线呈线性,线性范围为0.0625～10 ng/mL,R2=0.995,灵敏度IC50为1.45 ng/mL;其平均批内和批间变异系数均＜10％;除与二氟沙星的交叉反应率达到98.08％以外,与其他氟喹诺酮类药物的交叉反应率均＜8％,与其他非氟喹诺酮类药物均无交叉反应;SAR标准溶液的最低检测限(LOD)为0.32 ng/mL,SAR在鸡肉样品中的LOD为0.46 μg/kg;添加回收率在88.3％～106.7％范围内,其变异系数≤12.2％.结果表明,本研究建立的ic-CLEIA方法检测速度快、灵敏度高,适用于动物源性食品中SAR残留的大量检测,为SAR残留检测提供了新的方法.

外膜蛋白A(OmpA)为副溶血弧菌主要表面蛋白之一,具有较强的免疫原性,在疫苗上有很好的应用前景.为评价副溶血弧菌OmpA的免疫效果,本研究通过PCR方法获得副溶血弧菌SH112株的ompA(VP0764)基因的全长片段,测序结果显示该蛋白编码区全长990 bp.对其编码蛋白质进行生物信息学分析,结果显示该蛋白是含有信号肽的稳定蛋白,无跨膜结构,有11个抗原决定簇,具有较高免疫原性.扩增去除信号肽序列的成熟蛋白基因903 bp,并构建重组表达载体pET28a-ompA进行原核表达,结果显示,获得分子量约为39 ku的重组蛋白His-OmpA(VP0764).采用该蛋白免疫ICR小鼠(0.1 mg/只)3次后收集血清制备鼠多克隆抗血清,利用ELISA检测抗血清效价,结果显示制备的鼠多克隆抗血清效价达1:32000以上.利用western blot检测制备的鼠多克隆抗血清,结果显示该血清可与表达蛋白和全菌蛋白发生特异性反应,表明所表达的目的蛋白具有良好免疫原性,能识别天然OmpA蛋白.将45只8周龄ICR小鼠随机分成3组,分别免疫灭活的SH112全菌疫苗(1×108 cfu/只)、His-OmpA(VP0764)重组蛋白(0.1 mg/只)及PBS(200μL/只),每两周免疫一次,三免后10 d采用副溶血弧菌SH112株(2×107 cfu/只)攻毒,结果显示免疫SH112全菌和His-OmpA(VP0764)重组蛋白的实验组小鼠免疫保护率分别是73％和40％,与对照组差异显著.本研究为副溶血弧菌OmpA的功能与疫苗开发研究奠定了基础.